Journal: Cell Death & Disease

Article Title: Non-canonical NOTCH1 signaling regulates ferroptosis vulnerability in dormant lung cancer cells with stable resistance

doi: 10.1038/s41419-025-08355-9

Figure Lengend Snippet: A Establishment of in vitro cisplatin-resistant NSCLC models. The models were established according to the schematic diagram in the upper panel and validated with proliferation assays shown in the lower panel (n = 3 independent experiments). See cisplatin response assays for the IC50 of cisplatin in Fig. . B Representative images of autofluorescence in A549 and A549CR cells. Scale bar, 100 μm. C Representative brightfield and autofluorescence images of cisplatin-exposed A549CR cells cultured without passaging in cisplatin-free medium for 98 days. Colonies are indicated by dashed outlines. Scale bar, 50 μm. D Flow cytometric analysis of autofluorescence in A549/A549CR and HCC827/HCC827CR cells (n = 4 independent experiments). Results are summarized in Fig. . E Representative flow cytometric analysis and quantification of the riboflavin fluorescence in A549CR cells cultured in riboflavin-containing or -free (or R-free) medium for 28 days (n = 3 independent experiments). MFI, mean fluorescence intensity. The experimental design is shown in Fig. . F Flow cytometric analysis of NOTCH1 + 7-AAD - fractions in A549/A549CR and HCC827/HCC827CR cells, and in A549 and HCC287 clones with the sh NOTCH1-2 knockdown (n = 3 independent experiments). Results are summarized in Fig. . 7-AAD, 7-Aminoactinomycin D. G Flow cytometric analysis of the riboflavin + NOTCH1 + 7-AAD - population in both A549/A549CR and HCC827/HCC827CR cells (n = 3 independent experiments). Results are summarized in Fig. . H Representative flow cytometric analysis (left panel) and quantification (right panel) of the riboflavin + NOTCH1 + 7-AAD - (or R + N + ) population in clinical NSCLC specimens before and after therapy (n = 3 or 5 patients per group, Table ). The responsive group includes patients with a complete pathologic response or a major pathologic response, whereas the non-responsive group encompasses all other treated patients. SSC, side scatter. the data are presented as mean ± standard deviation (SD). P values were calculated using Student’s unpaired t -tests ( A ), or paired t -tests ( E ).

Article Snippet: The chemicals and compounds cisplatin (#HY-17394), RO4929097 (#HY-11102), PF-03084014 (#HY-15185B), Ferrostain-1 (# HY100579 ), Z-VAD-FMK (#HY16658B), FSEN1 (#HY-153629), BAI1 (#HY-103269), PEG300 (#HY-Y0873) and riboflavin (#HY-B0456) were obtained from MedChemExpress (Monmouth Junction, NJ).

Techniques: In Vitro, Cell Culture, Passaging, Fluorescence, Clone Assay, Knockdown, Standard Deviation

Journal: Cell Death & Disease

Article Title: Non-canonical NOTCH1 signaling regulates ferroptosis vulnerability in dormant lung cancer cells with stable resistance

doi: 10.1038/s41419-025-08355-9

Figure Lengend Snippet: A Schematic design for investigating the effect of cisplatin on the cisplatin-naïve and cisplatin-exposed riboflavin + NOTCH1 + populations derived from A549 and A549CR cells, respectively. B Representative Annexin V assays (left panel) and quantification (right panel) for the response of the cisplatin-naïve and cisplatin-exposed riboflavin + NOTCH1 + populations to cisplatin (n = 3 independent experiments). DMF (N, N-dimethylformamide) served as the diluent for cisplatin. C, D Assessment ( D ) of the irreversible chemoresistance of riboflavin + NOTCH1 + A549CR cells after 8 days of cisplatin-free culture (n = 3 independent experiments), according to the schematic design ( C ). Please also see representative Annexin V assays in Fig. . E CCK-8 assays for assessing the proliferation of 1 × 10 3 cisplatin-naïve and cisplatin-exposed riboflavin + NOTCH1 + cells sorted from the cell lines A549 and A549CR, respectively (n = 3 independent experiments). F Representative protein-flow analysis (left panel) and quantification (right panel) with an anti-Ki-67 antibody for the Ki-67 + cycling fractions in cisplatin-naïve and -exposed riboflavin + NOTCH1 + cells (n = 3 independent experiments). G Representative flow cytometric analyses (left panel) and quantification (right panel) of ROS levels in cisplatin-naïve and -exposed riboflavin + NOTCH1 + cells (n = 3 independent experiments). H Representative flow cytometric analysis (left panel) and quantification (right panel) of relative ROS levels in the riboflavin + NOTCH1 + or riboflavin - NOTCH1 - populations from NSCLC specimens treated with chemotherapy and immunotherapy (n = 3 independent experiments). I Representative confocal images of mitochondria in A549 and A549CR cells, which were stained with MitoTracker red CMXRos. The fluorescence of riboflavin is shown in green and that of mitochondria in red. R - N - riboflavin - NOTCH1 - ; R + N + , riboflavin + NOTCH1 + ; ZA, Zombie Aqua; scale bar, 5 µm; error bars, mean ± SD. P values were calculated using one way ANOVA with Tukey’s tests ( B , D – G ) or Student’s unpaired t -tests ( H ).

Article Snippet: The chemicals and compounds cisplatin (#HY-17394), RO4929097 (#HY-11102), PF-03084014 (#HY-15185B), Ferrostain-1 (# HY100579 ), Z-VAD-FMK (#HY16658B), FSEN1 (#HY-153629), BAI1 (#HY-103269), PEG300 (#HY-Y0873) and riboflavin (#HY-B0456) were obtained from MedChemExpress (Monmouth Junction, NJ).

Techniques: Derivative Assay, CCK-8 Assay, Staining, Fluorescence

Journal: Cell Death & Disease

Article Title: Non-canonical NOTCH1 signaling regulates ferroptosis vulnerability in dormant lung cancer cells with stable resistance

doi: 10.1038/s41419-025-08355-9

Figure Lengend Snippet: A enrichment of the chemoresistance program for A549CR cells, but not for A549 cells, was determined by GSEA with multiple chemoresistance signatures (n=3 independent experiments for Fig.3A-J, Table S2). B, C GSEA plots illustrated no enrichment of apoptosis ( B ) and ferroptosis ( C ) regulatory programs for A549CR cells and A549 cells. D GSEA demonstrated that the EMT program was correlated with A549 cells, but not with A549CR cells. E, F GSEA revealed that the p53 pathway ( E ) was positively enriched in A549CR cells, whereas the SenMayo senescence program ( F ) was not. G Gene ontology (GO) analysis of DEG revealed a downregulation of cell cycle programs in A549CR cells compared to A549 cells. H GSEA demonstrated that the cell cycle regulatory programs were inhibited in A549CR cells, but not in A549 cells. I GSEA plots showed a significant correlation of the G0 program with A549CR cells, but not with A549 cells. J Volcano plot illustrated the significantly upregulated (red) and downregulated (blue) genes in a comparison between A549CR and A549 cells. A dashed line represents the threshold of p < 0.0010 for the DEG. The top 10 DEG are labeled with their gene names. See also the top 10 DEG in Table . K Flow cytometric quantification of the CD82 + fractions in the cisplatin-naïve and cisplatin-exposed riboflavin + NOTCH1 + 7-AAD - cells (n = 3 independent experiments). See representative flow cytometric analyses in Fig. . L Flow cytometric quantification of the CD82 + fractions in the riboflavin - NOTCH1 - 7-AAD - and riboflavin + NOTCH1 + 7-AAD - cells from NSCLC specimens treated with immunotherapy and chemotherapy (n = 4 independent experiments). See representative flow cytometric analysis in Fig. . M Assessment of the mRNA levels of SLC52A1 , SLC52A2 and SLC52A3 in the RNA-seq dataset phs000178 derived from 174 NSCLC tumors. N RT-PCR analysis detected the mRNA levels of SLC52A1 , SLC52A2 , SLC52A3 in riboflavin + NOTCH1 + and riboflavin - NOTCH1 - cells sorted from the clinical NSCLC specimens LC012, LC013, and LC014 (n = 3 independent experiments). O Protein-flow analysis with an anti-SLC52A2 antibody detected SLC52A2 expression in riboflavin + NOTCH1 + ZA - and riboflavin - NOTCH1 - ZA - cells gated from the clinical NSCLC specimen LC011 (n = 1 independent experiment). R - N - , riboflavin - NOTCH1 - ; R + N + , riboflavin + NOTCH1 + ; ZA, Zombie Aqua; TPM, transcripts per million; error bars, mean ± SD. P values were calculated using one way ANOVA with Tukey’s tests ( K ) or Student’s unpaired t -tests ( L, N ). The adjusted P values were calculated using Wald tests with the Benjamini-Hochberg correction in DEseq2 ( M ).

Article Snippet: The chemicals and compounds cisplatin (#HY-17394), RO4929097 (#HY-11102), PF-03084014 (#HY-15185B), Ferrostain-1 (# HY100579 ), Z-VAD-FMK (#HY16658B), FSEN1 (#HY-153629), BAI1 (#HY-103269), PEG300 (#HY-Y0873) and riboflavin (#HY-B0456) were obtained from MedChemExpress (Monmouth Junction, NJ).

Techniques: Comparison, Labeling, RNA Sequencing, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Cell Death & Disease

Article Title: Non-canonical NOTCH1 signaling regulates ferroptosis vulnerability in dormant lung cancer cells with stable resistance

doi: 10.1038/s41419-025-08355-9

Figure Lengend Snippet: A GSEA of bulk RNA-seq data demonstrated that the canonical NOTCH pathway was not enriched in A549 and A549CR cells (n = 3 independent experiments). B Representative Western blot analysis (left panel) and quantification (right panel) of key regulators in the canonical NOTCH1 pathway (n = 3 independent experiments). FL, full-length NOTCH1; NTM, NOTCH1 transmembrane and intracellular region. C GSEA revealed the activation of cell cycle and EMT programs in A549CR cells in response to RO treatment. D Volcano plot showed the upregulated (red) and downregulated (blue) genes in RO- vs. dimethyl sulfoxide (DMSO)-treated A549CR cells. The dashed line represents the significance threshold of p < 0.0010. Please see a list of the top 10 DEG in Table . E STRING protein network analysis revealed protein-protein interactions between the proteins encoded by the top 20 upregulated (orange bubbles) and downregulated (green bubbles) genes in A549CR cells in response to RO treatment. The 3 groups of genes highlighted by dashed circles are associated with pro-EMT, pro-ferroptotic, and pro-vitamin-K/E-FSP1 functions. F Protein-flow analysis with the monoclonal antibody 6A7 for BAX activation in riboflavin + mCherry + ZA - SRCC gated from A549-shNC and A549- shNOTCH1 -2 cells (n = 3 independent experiments). G Protein-flow analysis with the monoclonal antibody 6A7 for BAX activation in riboflavin + mCherry + ZA - SRCC gated from A549- shNOTCH1-2 cells with the overexpression of flag-tagged NICD1 or mNICD1 (n = 3 independent experiments). Both A549- shNC and A549- shNOTCH1-2 were positive for mCherry. H Flow cytometric analysis for the fluorescence of LysoTracker Deep Red in riboflavin + mCherry + 7-AAD - SRCC gated from A549- shNOTCH1-2 cells with the overexpression of flag-tagged NICD1 or mNICD1 (n = 3 independent experiments). I Protein-flow analysis with the monoclonal antibody SY28-05 for AKT phosphorylation (S473) in riboflavin + mCherry + ZA - SRCC gated from A549- shNOTCH1-2 cells with the overexpression of flag-tagged NICD1 or mNICD1 (n = 2 independent experiments). Both A549-shNC and A549- shNOTCH1-2 are positive for mCherry. ZA, Zombie Aqua fluorescence for dead cells; RO, RO4929097; error bars, mean ± SD. P values were calculated using Student’s unpaired t -tests ( B ), Student’s paired t -tests ( F ), or one way ANOVA with Tukey’s tests ( H ).

Article Snippet: The chemicals and compounds cisplatin (#HY-17394), RO4929097 (#HY-11102), PF-03084014 (#HY-15185B), Ferrostain-1 (# HY100579 ), Z-VAD-FMK (#HY16658B), FSEN1 (#HY-153629), BAI1 (#HY-103269), PEG300 (#HY-Y0873) and riboflavin (#HY-B0456) were obtained from MedChemExpress (Monmouth Junction, NJ).

Techniques: RNA Sequencing, Western Blot, Activation Assay, Protein-Protein interactions, Over Expression, Fluorescence, Phospho-proteomics

Journal: Cell Death & Disease

Article Title: Non-canonical NOTCH1 signaling regulates ferroptosis vulnerability in dormant lung cancer cells with stable resistance

doi: 10.1038/s41419-025-08355-9

Figure Lengend Snippet: A Schematic design of the combinatorial treatment with GSI and cisplatin. B Viability of A549CR cells in response to cisplatin, RO, or both was assessed using CCK-8 assays (n = 3 independent experiments). C , D Representative Annexin V assay ( C ) and quantification ( D ) for riboflavin + 7-AAD − SRCC in response to cisplatin, RO, or both (n = 3 independent experiments). E Annexin V assays for the effect of cisplatin on riboflavin + 7-AAD - A549-shNC and A549- shNOTCH1-2 cells (n = 3 independent experiments). See also representative assays in Fig. . F Assessment of the absolute number of riboflavin + 7AAD − SRCC in xenograft tumors treated with RO alone, cisplatin alone, or both (n = 5–8 mice per group). G Assessment of the Annexin V - 7AAD - live cell fraction within the riboflavin + SRCC compartment derived from xenograft tumors treated with RO alone, cisplatin alone, or both (n = 5–8 mice per group). H Representative Annexin V assay for the effect of the ferroptosis inhibitor ferrostatin-1 (10 µmol/L), the apoptosis inhibitor Z-VAD-FMK (10 µmol/L), and the necroptosis inhibitor Nec-1s (10 µmol/L) on the death of riboflavin + 7-AAD - SRCC induced by the combinatorial treatment of RO and cisplatin (n = 2 independent experiments). I Protein-flow analysis with the monoclonal antibody 6A7 for BAX activation in riboflavin + ZA - A549CR cells in response to 6-, 12-, or 24-hour treatment with RO and cisplatin (n = 1–3 independent experiments). J , K Assessment of the lysosomal integrity of riboflavin + SRCC treated with RO and cisplatin or with RO, cisplatin, and BAI1 for 24 hours. The lysosomal integrity is determined using fluorescent microscopy ( J , n = 1 independent experiment) or flow cytometry ( K , n = 3 independent experiments) with the dye LysoTracker Deep Red. Scale bar, 50 μm; ZA, Zombie Aqua fluorescence for dead cells; RO, RO4929097; error bars, mean ± SD. P values were calculated by one way ANOVA with Tukey’s tests ( B , D , E , K ), Student’s unpaired t -tests ( F , G ) or Student’s paired t -tests ( I ).

Article Snippet: The chemicals and compounds cisplatin (#HY-17394), RO4929097 (#HY-11102), PF-03084014 (#HY-15185B), Ferrostain-1 (# HY100579 ), Z-VAD-FMK (#HY16658B), FSEN1 (#HY-153629), BAI1 (#HY-103269), PEG300 (#HY-Y0873) and riboflavin (#HY-B0456) were obtained from MedChemExpress (Monmouth Junction, NJ).

Techniques: CCK-8 Assay, Annexin V Assay, Derivative Assay, Activation Assay, Microscopy, Flow Cytometry, Fluorescence

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Biomimetic Remodeling of Microglial Riboflavin Metabolism Ameliorates Cognitive Impairment by Modulating Neuroinflammation.

doi: 10.1002/advs.202300180

Figure Lengend Snippet: Scheme 1. Schematic illustration showing the preparation and underlying mechanism of MNPs@FMN in memory protection. A) Preparation pro- cess of biomimetic nanocarriers (MNPs@FMN). B) Schematic diagram showing the proposed mechanism of MNPs@FMN crossing the BBB to become recruited by microglia. Briefly, MNPs@FMN penetrates the BBB via binding cell surface receptors on the brain endothelial cells. Afterward, MNPs@FMN accumulates in microglia, and released FMN inhibits RFK via KMT2B, while RFK promotes the TNFR1/NF-𝜅B signaling pathway. Ulti- mately, MNPs@FMN restores cognitive function by suppressing the inflammatory response.

Article Snippet: Anti-TNFR1 (H-5, sc8436) and RFK (E-7, sc-398830) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Binding Assay

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Biomimetic Remodeling of Microglial Riboflavin Metabolism Ameliorates Cognitive Impairment by Modulating Neuroinflammation.

doi: 10.1002/advs.202300180

Figure Lengend Snippet: Figure 2. Microglial RFK contributes to LPS-induced inflammation via the TNFR1/NF-𝜅B pathway. A-C) Representative blots and quantification showing RFK, TNFR1, p-NF-𝜅B, NF-𝜅B, p-I𝜅B𝛼and I𝜅B𝛼expression levels in LPS-treated BV2 cells with Control siRNA or Rfk siRNA n = 3 per group. D) Immunofluorescence staining and quantification of TNFR1 with the membrane dye DIL in LPS-treated BV2 cells with Control siRNA or Rfk siRNA n = 6–7. Scale bars, 20 μm. Magnified images are shown in the right columns. Scale bars, 5 μm. E,F) The mRNA expression levels of Il-1b, Il-6, Tnfa, Ifng, Cx3cr1, Tmem119, Csf1r, and P2ry12 in LPS-treated BV2 cells with Control siRNA or Rfk siRNA n = 3 per group. G) The secretion of IL-1𝛽, IL-6, and TNF-𝛼in the supernatants of LPS-treated BV2 cells with Control siRNA or Rfk siRNA n = 3 per group. H) The mRNA expression levels of Il-1b, Il-6, Tnfa, and Ifng in LPS-treated primary microglia with Control siRNA or Rfk siRNA n = 3 per group. I–K) Representative blots and quantification showing RFK, TNFR1, p-NF-𝜅B, NF-𝜅B, p-I𝜅B𝛼and I𝜅B𝛼expression levels in LPS-treated primary microglia with Control siRNA or Rfk siRNA n = 3 per group. L) Immunofluorescence staining and quantification of RFK with Iba1 in LPS-treated primary microglia with Control siRNA or Rfk siRNA n = 8–11. Scale bars, 40 μm. Magnified images are shown in the right columns. Scale bars, 13 μm. Results are expressed as mean ± SEM. **p < 0.01, *p < 0.05 versus Ctrl siRNA; ##p < 0.01, #p < 0.05 versus LPS + Ctrl siRNA. Statistical significance was determined using One-way ANOVA followed by Tukey’s post-hoc test.

Article Snippet: Anti-TNFR1 (H-5, sc8436) and RFK (E-7, sc-398830) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Control, Staining, Membrane, Expressing

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Biomimetic Remodeling of Microglial Riboflavin Metabolism Ameliorates Cognitive Impairment by Modulating Neuroinflammation.

doi: 10.1002/advs.202300180

Figure Lengend Snippet: Figure 3. FMN supplementation suppresses LPS-induced inflammation in BV2 cells and primary microglia. A) Schematic model of the riboflavin biosyn- thesis metabolic pathway. RFK, riboflavin kinase; FMN, flavin mononucleotide; FAD, flavin adenine dinucleotide. B-D) The supernatant levels of IL-1𝛽, IL-6, and TNF-𝛼in LPS-treated BV2 cell cultures with different doses of FMN or FAD n = 3 per group. E–G) Representative blots and quantification of RFK, TNFR1, p-NF-𝜅B, NF-𝜅B, p-I𝜅B𝛼, and I𝜅B𝛼expression levels in LPS-treated BV2 cells with 200 μM FMN. n = 3 per group. H,I) Immunofluorescence staining and quantification of TNFR1 with the membrane dye DIL in LPS-treated BV2 cells with FMN n = 6–8. Scale bars, 20 μm. Magnified images are shown in the right columns. Scale bars, 5 μm. (J) The mRNA expression levels of Il-1b, Il-6, and Tnfa in LPS-treated primary microglia with 200 μM FMN n = 3 per group. K-M) Representative blots and quantification of RFK, TNFR1, p-NF-𝜅B, NF-𝜅B, p-I𝜅B𝛼and I𝜅B𝛼expression levels in LPS-treated primary microglia with 200 μM FMN n = 3 per group. N,O) Immunofluorescence staining and quantification of RFK with Iba1 in LPS-treated primary microglia with 200 μM FMN n = 7–9. Scale bars, 40 μm. Magnified images are shown in the right columns. Scale bars, 13 μm. Results are expressed as mean ± SEM. **p < 0.01, *p < 0.05 versus Ctrl; ##p < 0.01, #p < 0.05 versus LPS. Statistical significance was determined using one-way ANOVA and Tukey’s tests for post hoc comparisons.

Article Snippet: Anti-TNFR1 (H-5, sc8436) and RFK (E-7, sc-398830) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Staining, Membrane, Expressing

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Biomimetic Remodeling of Microglial Riboflavin Metabolism Ameliorates Cognitive Impairment by Modulating Neuroinflammation.

doi: 10.1002/advs.202300180

Figure Lengend Snippet: Figure 4. FMN regulates RFK expression via KMT2B. Wild-type mice were intraperitoneally injected with 40 mg/kg FMN for 5 weeks, and the hippocampal samples were subjected for RNA-seq. A) Volcano plot showing the DEGs between FMN and Ctrl mice. B,C) Representative KEGG and GO pathways enriched by downregulated DEGs between FMN and Ctrl mice. D) DEGs enriched in the “Cytokine-mediated signaling pathway”, “Cell activation involved in immune response”, and “Positive regulation of cytokine production”. E,F) Representative blots and quantification showing KMT2B expression levels in Control and LPS-treated primary microglia n = 3 per group. G,H) Representative blots and quantification showing KMT2B expression levels in the hippocampus of Control mice and the LPS mouse model n = 4 per group. (I) Kmt2b mRNA expression levels in the hippocampus of WT mice and the 5xFAD mouse model. n = 3 per group. J,K) Representative blots and quantification showing KMT2B expression levels in the hippocampus of WT mice and the 5xFAD mouse model n = 4 per group. L,M) Representative blots and quantification showing KMT2B and RFK expression levels in LPS-treated primary microglia with Control siRNA or Kmt2b siRNA n = 3 per group. N–P) Representative blots and quantification of KMT2B, RFK, TNFR1, p-NF-𝜅B, and NF-𝜅B expression levels in LPS-treated primary microglia with FMN and KMT2B recombinant protein n = 3 per group. Q) The mRNA expression levels of Il-1b, Il-6, Tnfa, and Ifng in LPS-treated primary microglia with FMN and KMT2B recombinant protein n = 3 per group. Results are expressed as mean ± SEM. **p < 0.01, *p < 0.05 versus Ctrl or Ctrl siRNA; ##p < 0.01, #p < 0.05 versus LPS or LPS + Ctrl siRNA; &&p < 0.01, &p < 0.05 versus LPS + FMN. Statistical significance was determined using Student’s t-test (for E-K) and one-way ANOVA and Tukey’s tests for post hoc comparisons (for L-Q).

Article Snippet: Anti-TNFR1 (H-5, sc8436) and RFK (E-7, sc-398830) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Expressing, Injection, RNA Sequencing, Activation Assay, Control, Recombinant

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Biomimetic Remodeling of Microglial Riboflavin Metabolism Ameliorates Cognitive Impairment by Modulating Neuroinflammation.

doi: 10.1002/advs.202300180

Figure Lengend Snippet: Figure 5. Hippocampal Rfk knockdown attenuates LPS-induced cognitive impairment and inflammatory response. A) Experimental design for Rfk knock- down and FMN administration in the LPS-induced mouse model. B) Travel path tracings of mice in the Y-maze test. C) Spontaneous alterations of LPS mice treated with LV-Rfk shRNA or FMN in the Y-maze. D) Representative path tracings in each quadrant during the probe trial. E-H) The escape latency over a five-day training course (panel E), latency in the probe test (panel F), time spent in the target zone (panel G), and number of target crossings (panel H) in the Morris water maze n = 7–8. I-K) Representative blots and quantification of RFK, TNFR1, p-NF-𝜅B, NF-𝜅B, p-I𝜅B𝛼and I𝜅B𝛼expression levels in the hippocampus of LPS mice treated with LV-Rfk shRNA or FMN n = 3 per group. L) Immunofluorescence staining and quantification of Iba1-positive cells in the hippocampus of LPS mice treated with LV-Rfk shRNA or FMN. Scale bars, 40 μm. Magnified images and skeletal diagrams of Iba1-positive cells are shown to the right of each staining image. Scale bars, 10 μm n = 8–9. M,N) The mRNA expression levels of Il-1b, Il-6, Tnfa, Tmem119, Cx3cr1, Csf1r, and P2ry12 in the hippocampus of LPS mice treated with LV-Rfk shRNA or FMN n = 3 per group. Results are expressed as mean ± SEM. **p < 0.01, *p < 0.05 versus WT; ##p < 0.01, #p < 0.05 versus LPS; &&p < 0.01, &p < 0.05 versus LPS + Rfk shRNA. Statistical significance was determined using one-way ANOVA and Tukey’s tests for post hoc comparisons.

Article Snippet: Anti-TNFR1 (H-5, sc8436) and RFK (E-7, sc-398830) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Knockdown, shRNA, Staining, Expressing

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Biomimetic Remodeling of Microglial Riboflavin Metabolism Ameliorates Cognitive Impairment by Modulating Neuroinflammation.

doi: 10.1002/advs.202300180

Figure Lengend Snippet: Figure 11. MNPs@FMN improves cognitive function and reduces pro-inflammatory response in the 5xFAD mouse model. A) Travel path tracings of mice in the Y maze test. B) Spontaneous alterations of 5xFAD mouse intravenous delivery of PBS, free FMN, or MNPs@FMN in the Y-maze. C) Representative path tracings in each quadrant during the probe trial. D–F) The escape latency over a five-day training course (panel D), latency in the probe test (panel E), and time spent in the target zone (panel F) in the Morris water maze. n = 6–7. G) The mRNA expression levels of Il-1b, Il-6, Tnfa, Tmem119, Cx3cr1, Csf1r, and P2ry12 in the hippocampi of 5xFAD mice after intravenous delivery of PBS, free FMN or MNPs@FMN. n = 3 per group. H–J) Representative blots and quantification of KMT2B, RFK, TNFR1, p-NF-𝜅B, NF-𝜅B, p-I𝜅B𝛼and I𝜅B𝛼expression levels in the hippocampi of 5xFAD mice after intravenous delivery of PBS, free FMN or MNPs@FMN. n = 3 per group. K) Immunofluorescence staining of A𝛽plaque (6E10) with Iba1

Article Snippet: Anti-TNFR1 (H-5, sc8436) and RFK (E-7, sc-398830) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Expressing, Staining